Simultaneous Proteoform Analysis of Histones H3 and H4 with a Simplified Middle-Down Proteomics Method.

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer "middle-down" protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis.

Label-free proteome profiling reveals developmental-dependent patterns in young barley grains.

UNLABELLED: Due to its importance as a cereal crop worldwide, high interest in the determination of factors influencing barley grain quality exists. This study focusses on the elucidation of protein networks affecting early grain developmental processes. NanoLC-based separation coupled to label-free MS detection was applied to gain insights into biochemical processes during five different grain developmental phases (pre-storage until storage phase, 3days to 16days after flowering). Multivariate statistics revealed two distinct developmental patterns during the analysed grain developmental phases: proteins showed either highest abundance in the middle phase of development - in the transition phase - or at later developmental stages - within the storage phase. Verification of developmental patterns observed by proteomic analysis was done by applying hypothesis-driven approaches, namely Western Blot analysis and enzyme assays. High general metabolic activity of the grain with regard to protein synthesis, cell cycle regulation, defence against oxidative stress, and energy production via photosynthesis was observed in the transition phase. Proteins upregulated in the storage phase are related towards storage protein accumulation, and interestingly to the defence of storage reserves against pathogens. A mixed regulatory pattern for most enzymes detected in our study points to regulatory mechanisms at the level of protein isoforms. BIOLOGICAL SIGNIFICANCE: In-depth understanding of early grain developmental processes of cereal caryopses is of high importance as they influence final grain weight and quality. Our knowledge about these processes is still limited, especially on proteome level. To identify key mechanisms in early barley grain development, a label-free data-independent proteomics acquisition approach has been applied. Our data clearly show, that proteins either exhibit highest expression during cellularization and the switch to the storage phase (transition phase, 5-7 DAF), or during storage product accumulation (10-16 DAF). The results highlight versatile cellular metabolic activity in the transition phase and strong convergence towards storage product accumulation in the storage phase. Notably, both phases are characterized by particular protective mechanism, such as scavenging of oxidative stress and defence against pathogens, during the transition and the storage phase, respectively.

Spatiotemporal profiling of starch biosynthesis and degradation in the developing barley grain.

Barley (Hordeum vulgare) grains synthesize starch as the main storage compound. However, some starch is degraded already during caryopsis development. We studied temporal and spatial expression patterns of genes coding for enzymes of starch synthesis and degradation. These profiles coupled with measurements of selected enzyme activities and metabolites have allowed us to propose a role for starch degradation in maternal and filial tissues of developing grains. Early maternal pericarp functions as a major short-term starch storage tissue, possibly ensuring sink strength of the young caryopsis. Gene expression patterns and enzyme activities suggest two different pathways for starch degradation in maternal tissues. One pathway possibly occurs via alpha-amylases 1 and 4 and beta-amylase 1 in pericarp, nucellus, and nucellar projection, tissues that undergo programmed cell death. Another pathway is deducted for living pericarp and chlorenchyma cells, where transient starch breakdown correlates with expression of chloroplast-localized beta-amylases 5, 6, and 7, glucan, water dikinase 1, phosphoglucan, water dikinase, isoamylase 3, and disproportionating enzyme. The suite of genes involved in starch synthesis in filial starchy endosperm is much more complex than in pericarp and involves several endosperm-specific genes. Transient starch turnover occurs in transfer cells, ensuring the maintenance of sink strength in filial tissues and the reallocation of sugars into more proximal regions of the starchy endosperm. Starch is temporally accumulated also in aleurone cells, where it is degraded during the seed filling period, to be replaced by storage proteins and lipids.